Nemorubicin metabolite and analog reagents, antibody-drug conjugates and methods

ABSTRACT

The present invention relates to antibody-drug conjugate compounds of Formula I:
 
Ab-(L-D) p   I
 
     where one or more nemorubicin metabolite or analog drug moieties (D) are covalently attached by a linker (L) to an antibody (Ab) which binds to one or more tumor-associated antigens or cell-surface receptors. These compounds may be useful in methods of diagnosis or treatment of cancer, and other diseases and disorders.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 12/865,354, filedNov. 30, 2010, which is a National Stage application of InternationalApplication No. PCT/US2009/031199, filed Jan. 16, 2009, which claimspriority under 35 USC 119(e) to U.S. Provisional Ser. No. 61/025,504filed on Feb. 1, 2008, the entire contents of which are incorporated byreference.

FIELD OF THE INVENTION

The invention relates generally to compounds with anti-cancer activityand more specifically to antibodies conjugated with chemotherapeuticnemorubicin metabolite and analog drugs. The invention also relates tomethods of using the antibody-drug conjugate compounds for in vitro, insitu, and in vivo diagnosis or treatment of mammalian cells, orassociated pathological conditions.

BACKGROUND OF THE INVENTION

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders (Carter, P.(2006) Nature Reviews Immunology 6:343-357). The use of antibody-drugconjugates (ADC), i.e. immunoconjugates, for the local delivery ofcytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumorcells in the treatment of cancer, targets delivery of the drug moiety totumors, and intracellular accumulation therein, whereas systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Xie et al (2006) Expert. Opin. Biol.Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res. 66(6):3214-3121; Lawet al (2006) Cancer Res. 66(4):2328-2337; Wu et al (2005) NatureBiotech. 23(9):1137-1145; Lambert J. (2005) Current Opin. in Pharmacol.5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103;Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) CancerImmunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) AnticancerResearch 19:605-614). Maximal efficacy with minimal toxicity is soughtthereby. Efforts to design and refine ADC have focused on theselectivity of monoclonal antibodies (mAbs) as well as drug mechanism ofaction, drug-linking, drug/antibody ratio (loading), and drug-releasingproperties (McDonagh (2006) Protein Eng. Design & Sel.; Doronina et al(2006) Bioconj. Chem. 17:114-124; Erickson et al (2006) Cancer Res.66(8):1-8; Sanderson et al (2005) Clin. Cancer Res. 11:843-852; Jeffreyet al (2005) J. Med. Chem. 48:1344-1358; Hamblett et al (2004) Clin.Cancer Res. 10:7063-7070). Drug moieties may impart their cytotoxic andcytostatic effects by mechanisms including tubulin binding, DNA binding,or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibodies or protein receptorligands.

The anthracycline analog, doxorubicin (ADRIAMYCIN) is thought tointeract with DNA by intercalation and inhibition of the progression ofthe enzyme topoisomerase II, which unwinds DNA for transcription.Doxorubicin stabilizes the topoisomerase II complex after it has brokenthe DNA chain for replication, preventing the DNA double helix frombeing resealed and thereby stopping the process of replication.Doxorubicin and daunorubicin (DAUNOMYCIN) are prototype cytotoxicnatural product anthracycline chemotherapeutics (Sessa et al (2007)Cardiovasc. Toxicol. 7:75-79). Immunoconjugates and prodrugs ofdaunorubicin and doxorubicin have been prepared and studied (Kratz et al(2006) Current Med. Chem. 13:477-523; Jeffrey et al (2006) Bioorganic &Med. Chem. Letters 16:358-362; Torgov et al (2005) Bioconj. Chem.16:717-721; Nagy et al (2000) Proc. Natl. Acad. Sci. 97:829-834;Dubowchik et al (2002) Bioorg. & Med. Chem. Letters 12:1529-1532; Kinget al (2002) J. Med. Chem. 45:4336-4343; U.S. Pat. No. 6,630,579). Theantibody-drug conjugate BR96-doxorubicin reacts specifically with thetumor-associated antigen Lewis-Y and has been evaluated in phase I andII studies (Saleh et al (2000) J. Clin. Oncology 18:2282-2292; Ajani etal (2000) Cancer Jour. 6:78-81; Tolcher et al (1999) J. Clin. Oncology17:478-484).

Morpholino analogs of doxorubicin and daunorubicin, formed bycyclization on the glycoside amino group, have greater potency (Acton etal (1984) J. Med. Chem. 638-645; U.S. Pat. Nos. 4,464,529; 4,672,057;5,304,687). Nemorubicin is a semisynthetic analog of doxorubicin with a2-methoxymorpholino group on the glycoside amino of doxorubicin and hasbeen under clinical evaluation (Grandi et al (1990) Cancer Treat. Rew.17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phaseII/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedingsof the American Society for Clinical Oncology 22, Abs1448; Quintieri(2003) Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology24:14116)

Nemorubicin is named as(8S,10S)-6,8,11-trihydroxy-10-((2R,4S,5S,6S)-5-hydroxy-4-((S)-2-methoxymorpholino)-6-methyltetrahydro-2H-pyran-2-yloxy)-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,with CAS Reg. No. 108852-90-0, and has the structure:

Several metabolites of nemorubicin (MMDX) from liver microsomes havebeen characterized, including PNU(159682), (Quintieri et al (2005)Clinical Cancer Research, 11(4):1608-1617; Beulz-Riche et al (2001)Fundamental & Clinical Pharmacology, 15(6):373-378; EP 0889898; WO2004/082689; WO 2004/082579). PNU(159682) was remarkably more cytotoxicthan nemorubicin and doxorubicin in vitro, and was effective in vivotumor models. PNU(159682) is named as3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3″(R)-oxy-4″-morpholinyl]doxorubicin,and has the structure:

SUMMARY

The present invention provides nemorubicin metabolite and analog drugmoiety reagents for the preparation of therapeutic antibody-drugconjugate (ADC) compounds.

The present invention also provides nemorubicin metabolite and analogdrug-linker reagents for the preparation of therapeutic antibody-drugconjugate (ADC) compounds.

The present invention also provides therapeutic antibody-drug conjugate(ADC) compounds comprising nemorubicin metabolite and analog drugmoieties, with biological activity against cancer cells. The compoundsmay inhibit tumor growth in mammals and may be useful for treating humancancer patients.

Aspects of the invention include methods of making, methods ofpreparing, methods of synthesis, methods of conjugation, and methods ofpurification of the drug moiety reagents, the drug-linker reagents, andthe antibody-drug conjugate compounds.

Antibody-drug conjugate (ADC) compounds of the invention comprise anantibody (Ab) covalently attached by a linker (L) to one or morenemorubicin metabolite or analog drug moieties (D). The ADC may berepresented by Formula I:Ab-(L-D)_(p)  I

where one or more nemorubicin analog drug moieties (D) have thestructure:

wherein:

Y is N—X⁶ or O;

L is attached at one of X¹, X², X³, X⁴, X⁵, or X⁶; and

p is 1, 2, 3, 4, 5, 6, 7, or 8.

Another aspect of the invention is a composition comprising a mixture ofantibody-drug compounds of Formula I where the average drug loading perantibody is about 2 to about 5, or about 3 to about 4.

Another aspect of the invention is a pharmaceutical compositionincluding a Formula I ADC compound, a mixture of Formula I ADCcompounds, or a pharmaceutically acceptable salt or solvate thereof, anda pharmaceutically acceptable diluent, carrier, or excipient.

Another aspect provides a pharmaceutical combination comprising aFormula I ADC compound and a second compound having anticancerproperties or other therapeutic effects.

Another aspect is a method for killing or inhibiting the proliferationof tumor cells or cancer cells comprising treating the cells with anamount of an antibody-drug conjugate of Formula I, or a pharmaceuticallyacceptable salt or solvate thereof, being effective to kill or inhibitthe proliferation of the tumor cells or cancer cells.

Another aspect is a method of treating cancer comprising administeringto a patient a therapeutically effective amount of a pharmaceuticalcomposition including a Formula I ADC.

Another aspect includes articles of manufacture, i.e. kits, comprisingan antibody-drug conjugate, a container, and a package insert or labelindicating a treatment.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims. One skilled in theart will recognize many methods and materials similar or equivalent tothose described herein, which could be used in the practice of thepresent invention. The present invention is in no way limited to themethods and materials described. Unless defined otherwise, technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs, and are consistent with: Singleton et al., (1994) Dictionary ofMicrobiology and Molecular Biology, 2nd Ed., J. Wiley & Sons, New York,N.Y.; and Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York.

Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings.

When trade names are used herein, applicants intend to independentlyinclude the trade name product formulation, the generic drug, and theactive pharmaceutical ingredient(s) of the trade name product.

The term “amino acid side chain” includes those groups found in: (i)naturally occurring amino acids such as alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; (ii) minor amino acids suchas ornithine and citrulline; (iii) unnatural amino acids, beta-aminoacids, synthetic analogs and derivatives of naturally occurring aminoacids; and (iv) all enantiomers, diastereomers, isomerically enriched,isotopically labelled, protected forms, and racemic mixtures thereof.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Theimmunoglobulins can be derived from any species, including human,murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g., OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

An “ErbB receptor” is a receptor protein tyrosine kinase which belongsto the ErbB receptor family which are important mediators of cellgrowth, differentiation and survival. The ErbB receptor family includesfour distinct members including epidermal growth factor receptor (EGFR,ErbB1, HER1), HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) and HER4 (ErbB4or tyro2). The ErbB receptor will generally comprise an extracellulardomain, which may bind an ErbB ligand; a lipophilic transmembranedomain; a conserved intracellular tyrosine kinase domain; and acarboxyl-terminal signaling domain harboring several tyrosine residueswhich can be phosphorylated. The ErbB receptor may be a “nativesequence” ErbB receptor or an “amino acid sequence variant” thereof. TheErbB receptor may be native sequence human ErbB receptor. Accordingly, a“member of the ErbB receptor family” is EGFR (ErbB1), ErbB2, ErbB3,ErbB4 or any other ErbB receptor currently known or to be identified inthe future. Sequence identity screening has resulted in theidentification of two other ErbB receptor family members; ErbB3 (U.S.Pat. Nos. 5,183,884; 5,480,968; Kraus et al (1989) PNAS (USA)86:9193-9197) and ErbB4 (EP 599274; Plowman et al (1993) Proc. Natl.Acad. Sci. USA, 90:1746-1750; and Plowman et al (1993) Nature366:473-475). Both of these receptors display increased expression on atleast some breast cancer cell lines. Anti-ErbB2 antibodies have beencharacterized (U.S. Pat. Nos. 5,677,171; 5,821,337; 6,054,297;6,165,464; 6,407,213; 6,719,971; 6,800,738; Fendly et al (1990) CancerResearch 50:1550-1558; Kotts et al. (1990) In Vitro 26(3):59A; Sarup etal. (1991) Growth Regulation 1:72-82; Shepard et al. J. (1991) Clin.Immunol. 11(3):117-127; Kumar et al. (1991) Mol. Cell. Biol.11(2):979-986; Lewis et al. (1993) Cancer Immunol. Immunother.37:255-263; Pietras et al. (1994) Oncogene 9:1829-1838; Vitetta et al.(1994) Cancer Research 54:5301-5309; Sliwkowski et al. (1994) J. Biol.Chem. 269(20):14661-14665; Scott et al. (1991) J. Biol. Chem.266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994) 91:7202-7206;Lewis et al. (1996) Cancer Research 56:1457-1465; and Schaefer et al.(1997) Oncogene 15:1385-1394.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all, or substantially all, ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al (1986) Nature, 321:522-525; Riechmann et al(1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol.,2:593-596). Humanized anti-ErbB2 antibodies include huMAb4D5-1,huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7and huMAb4D5-8 (HERCEPTIN®, trastuzumab) as described in Table 3 of U.S.Pat. No. 5,821,337 expressly incorporated herein by reference; humanized520C9 (WO 93/21319) and humanized 2C4 antibodies.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

A “disorder” is any condition that would benefit from treatment of thepresent invention. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include benign and malignant tumors; leukemia andlymphoid malignancies, in particular breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic,prostate or bladder cancer; neuronal, glial, astrocytal, hypothalamicand other glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders. Anexemplary disorder to be treated in accordance with the presentinvention is a solid, malignant tumor.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may: (i)reduce the number of cancer cells; (ii) reduce the tumor size; (iii)inhibit, retard, slow to some extent and preferably stop cancer cellinfiltration into peripheral organs; (iv) inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; (v) inhibit tumor growth;and/or (vi) relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the drug may prevent growthand/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. In animal models, efficacy may be assessed by physicalmeasurements of the tumor during the course following administration ofthe ADC, and by determining partial and complete remission of tumor. Forcancer therapy, efficacy can, for example, be measured by assessing thetime to disease progression (TTP) and/or determining the response rate(RR).

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, gastrointestinal stromal tumor (GIST),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, colorectal cancer, endometrial or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, aswell as head and neck cancer.

An “ErbB-expressing cancer” is one comprising cells which have ErbBprotein present at their cell surface. An “ErbB2-expressing cancer” isone which produces sufficient levels of ErbB2 at the surface of cellsthereof, such that an anti-ErbB2 antibody can bind thereto and have atherapeutic effect with respect to the cancer.

A cancer which “overexpresses” a receptor, e.g. an ErbB receptor, is onewhich has significantly higher levels of the receptor, such as ErbB2, atthe cell surface thereof, compared to a noncancerous cell of the sametissue type. Such overexpression may be caused by gene amplification orby increased transcription or translation. Receptor overexpression maybe determined in a diagnostic or prognostic assay by evaluatingincreased levels of the receptor protein present on the surface of acell (e.g., via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of receptor-encoding nucleic acidin the cell, e.g., via fluorescent in situ hybridization (FISH; see WO98/45479), southern blotting, or polymerase chain reaction (PCR)techniques, such as real time quantitative PCR (RT-PCR). Overexpressionof the receptor ligand, may be determined diagnostically by evaluatinglevels of the ligand (or nucleic acid encoding it) in the patient, e.g.,in a tumor biopsy or by various diagnostic assays such as the IHC, FISH,southern blotting, PCR or in vivo assays described above. One may alsostudy receptor overexpression by measuring a shed antigen (e.g., ErbBextracellular domain) in a biological fluid such as serum (see, e.g.,U.S. Pat. No. 4,933,294; WO 91/05264; U.S. Pat. No. 5,401,638; and Siaset al (1990) J. Immunol. Methods 132: 73-80). Aside from the aboveassays, various other in vivo assays are available to the skilledpractitioner. For example, one may expose cells within the body of thepatient to an antibody which is optionally labeled with a detectablelabel, e.g., a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g., by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, andradioactive isotopes of Lu), chemotherapeutic agents, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including synthetic analogs andderivatives thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkyatingagents, antimetabolites, spindle poison plant alkaloids,cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CASNo. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifamib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the PI3K inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

“Alkyl” is C₁-C₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples of alkyl radicals include, but not limitedto: methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl,—CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu,n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl,—CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 20 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen, phosphorusand sulfur, the remaining ring atoms being C, where one or more ringatoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocyclesare described in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring carbon atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted independently with one or more substituentsdescribed herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

“Linker” or “link” means a chemical moiety comprising a covalent bond ora chain of atoms that covalently attaches an antibody to a drug moiety.In various embodiments of Formula I, a linker is specified as L. Linkerembodiments include divalent radical defined herein as Y^(1-x).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or l meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of an ADC.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” refers to an association of one ormore solvent molecules and an ADC. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

Nemorubicin Metabolite and Analog Drug Moieties

The nemorubicin metabolite or analog drug moieties of the invention havethe structure:

wherein Y is N—X⁶ or O; and the divalent linker, L, is attached at oneof X¹, X², X³, X⁴, X⁵, or X⁶. The linker is also covalently attached tothe antibody according to Formula I antibody-drug conjugate.

The nemorubicin metabolite and analog drug moieties D include allstereoisomers, including enantiomers, diastereomers, atropisomers, andracemic mixtures, i.e. any combination of R and S configurations at thechiral carbons of D.

Drug Moiety Reagents

The nemorubicin metabolite and analog drug moiety reagents have thestructure:

wherein Y is N—X⁶ or O; and

one of Z¹, Z², Z³, Z⁴, Z⁵, or Z⁶ comprises a reactive functional groupselected from maleimide, thiol, amino, alkyl bromide, alkyl iodide,carboxyl, and NHS ester.

Embodiments of the reactive functional group include NHR¹⁰, OH, SH,—CH₂CH₂SH, —CO₂H, and

where R¹⁰ is H, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, or C₁-C₂₀ heteroaryl,optionally substituted with one or more groups independently selectedfrom F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃, —CO₂H, —CONH₂, —CONHCH₃,—NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH, —OCH₃, —OCH₂CH₃, —S(O)₂NH₂,and —S(O)₂CH₃.

Accordingly, drug moiety reagents include the structures:

wherein Z¹, Z², Z³, Z⁴, Z⁵, and Z⁶ comprise a reactive functional groupselected from maleimide, thiol, amino, alkyl bromide, alkyl iodide,alkyl thiol, alkyl hydroxyl, alkyl amino, hydroxyl, carboxyl, and NHSester; or protected form of the functional group thereof.

The nemorubicin metabolite and analog drug moiety reagents may beprepared by cyclization of doxorubicin with2-iodo-1-(2-iodoethoxy)-1-methoxyethane, following the methods in U.S.Pat. No. 5,304,687 and WO 2005/005455. Cyclization of doxorubicin withfunctionalized versions of 2-iodo-1-(2-iodoethoxy)-1-methoxyethane allowfunctionalized nemorubicin analogs:

Bridged oxygen derivatives having the3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3″(R)-oxy-4″-morpholinyl]substructureof PNU(159682) may be prepared by reductive alkylation of doxorubicinwith functionalized analogs of the dialdehyde, 2,2′-oxydiacetaldehyde(U.S. Pat. Nos. 4,826,964; 4,672,057; 6,630,579).

Nemorubicin and other morpholino doxorubicin analogs may also becyclized to the3′-deamino-3″,4′-anhydro-[2″(S)-methoxy-3″(R)-oxy-4″-morpholinyl]substructureof PNA(159682)-type compounds by forming the N-oxide with hydrogenperoxide (GB 2296495) and oxidative cyclization with ferrous chlorideand an iron-complexing agent, such as tartaric acid (EP 0889898).

Embodiments of drug moiety reagents include the compounds:

Drug-Linker Reagents

Drug-linker reagents have the structure:

wherein Y is N—X⁶, S, or O; and

one of X¹, X², X³, X⁴, X⁵, or X⁶ comprises a linker and a reactivefunctional group selected from a maleimide group, a thiol group, acarboxyl group, and an NHS ester.

Accordingly, drug-linker reagents include the structures:

Linkers

The linker, L, attaches the antibody to a drug moiety through covalentbond(s). The linker is a bifunctional or multifunctional moiety whichcan be used to link one or more drug moiety (D) and an antibody unit(Ab) to form antibody-drug conjugates (ADC) of Formula I. The linker (L)may be stable outside a cell, i.e. extracellular, or it may be cleavableby enzymatic activity, hydrolysis, or other metabolic conditions.Antibody-drug conjugates (ADC) can be conveniently prepared using alinker having reactive functionality for binding to the drug moiety andto the antibody. A cysteine thiol, or an amine, e.g. N-terminus or aminoacid side chain such as lysine, of the antibody (Ab) can form a bondwith a functional group of a linker reagent, drug moiety (D) ordrug-linker reagent (D-L)

Many positions on nemorubicin metabolite and analog compounds may beuseful as the linkage position, depending upon the type of linkage. Forexample, ester linkages may be formed from a hydroxyl group on the drugmoiety; ketal and hydrazone linkages may be formed from a carbonyl groupon the drug moiety; amide, carbamate, and urea linkages may be formedfrom an amino group on the drug moiety; and various alkyl, ether,thioether, disulfide, and acyl linkages may be formed from the phenyland aryl rings on the drug moiety by Friedel-Crafts type alkylation andacylation reactions.

The linkers are preferably stable extracellularly. Before transport ordelivery into a cell, the antibody-drug conjugate (ADC) is preferablystable and remains intact, i.e. the antibody remains linked to the drugmoiety. The linkers are stable outside the target cell and may becleaved at some efficacious rate inside the cell. An effective linkerwill: (i) maintain the specific binding properties of the antibody; (ii)allow intracellular delivery of the conjugate or drug moiety; (iii)remain stable and intact, i.e. not cleaved, until the conjugate has beendelivered or transported to its targetted site; and (iv) maintain acytotoxic, cell-killing effect or a cytostatic effect of the nemorubicinmetabolite and analog drug moiety. Stability of the ADC may be measuredby standard analytical techniques such as mass spectroscopy, HPLC, andthe separation/analysis technique LC/MS.

Covalent attachment of the antibody and the drug moiety requires thelinker to have two reactive functional groups, i.e. bivalency in areactive sense. Bivalent linker reagents which are useful to attach twoor more functional or biologically active moieties, such as peptides,nucleic acids, drugs, toxins, antibodies, haptens, and reporter groupsare known, and methods have been described their resulting conjugates(Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: NewYork, p 234-242).

In another embodiment, the linker may be substituted with groups whichmodulate solubility or reactivity. For example, a sulfonate substituentmay increase water solubility of the reagent and facilitate the couplingreaction of the linker reagent with the antibody or the drug moiety, orfacilitate the coupling reaction of Ab-L with D, or D-L with Ab,depending on the synthetic route employed to prepare the ADC.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). US 2007/0092940 teachesengineering antibodies by introduction of reactive cysteine amino acids.

In some embodiments, a Linker has a reactive nucleophilic group which isreactive with an electrophilic group present on an antibody. Usefulelectrophilic groups on an antibody include, but are not limited to,aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilicgroup of a Linker can react with an electrophilic group on an antibodyand form a covalent bond to an antibody unit. Useful nucleophilic groupson a Linker include, but are not limited to, hydrazide, oxime, amino,hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide. The electrophilic group on an antibody provides aconvenient site for attachment to a Linker.

Nucleophilic groups on a drug moiety include, but are not limited to:amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide groups capable of reacting toform covalent bonds with electrophilic groups on linker moieties andlinker reagents including: (i) active esters such as NHS esters, HOBtesters, haloformates, and acid halides; (ii) alkyl and benzyl halidessuch as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups.

Linkers can be peptidic, comprising one or more amino acid units.Peptide linker reagents may be prepared by solid phase or liquid phasesynthesis methods (E. Schröder and K. Lübke, The Peptides, volume 1, pp76-136 (1965) Academic Press) that are well known in the field ofpeptide chemistry, including t-BOC chemistry (Geiser et al “Automationof solid-phase peptide synthesis” in Macromolecular Sequencing andSynthesis, Alan R. Liss, Inc., 1988, pp. 199-218) and Fmoc/HBTUchemistry (Fields, G. and Noble, R. (1990) “Solid phase peptidesynthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids”, Int. J.Peptide Protein Res. 35:161-214), on an automated synthesizer such asthe Rainin Symphony Peptide Synthesizer (Protein Technologies, Inc.,Tucson, Ariz.), or Model 433 (Applied Biosystems, Foster City, Calif.).

Exemplary amino acid linkers include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Amino acid side chains include those occurring naturally, as well asminor amino acids and non-naturally occurring amino acid analogs, suchas citrulline. Amino acid side chains include hydrogen, methyl,isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, as well as thefollowing structures:

When the amino acid side chains include is other than hydrogen(glycine), the carbon atom to which the amino acid side chain isattached is chiral. Each carbon atom to which the amino acid side chainis attached is independently in the (S) or (R) configuration, or aracemic mixture. Drug-linker reagents may thus be enantiomerically pure,racemic, or diastereomeric.

In exemplary embodiments, amino acid side chains are selected from thoseof natural and non-natural amino acids, including alanine,2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, norleucine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,γ-aminobutyric acid, α,α-dimethyl γ-aminobutyric acid, β,β-dimethylγ-aminobutyric acid, ornithine, and citrulline (Cit).

Embodiments of drug-linker reagents include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

R¹ and R² are independently an amino acid side chain selected fromhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the following structures:

R³ is H, C₁-C₈ alkyl, NR¹⁰C(O)R¹⁰, or C(O)CH₃;

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃; and

n is 0, 1, 2, 3, 4, 5, or 6.

Embodiments of drug-linker reagents include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, O—(C₁-C₆ alkyl)-NR¹⁰, O(C₁-C₆ alkyl)O, orOC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰;

R¹ and R² are independently an amino acid side chain selected fromhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the structures:

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃; and

n is independently 0, 1, 2, 3, 4, 5, or 6.

Embodiments of drug-linker reagents include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, OC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰; and

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

Embodiments of drug-linker reagents include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, OC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰;

Y³ is (C(R¹⁰)₂)_(r);

R¹ and R² are independently an amino acid side chain selected fromhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the structures:

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6;

r is 0, 1, 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of drug-linker reagents include:

Embodiments of drug-linker reagents include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, OC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰;

Y³ is (C(R¹⁰)₂)_(r);

R¹ is independently an amino acid side chain selected from hydrogen,methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, and thestructures:

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6;

r is 0, 1, 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of drug-linker reagents include:

wherein

Y³ is (C(R¹⁰)₂)_(r);

Y⁴ is O, NR¹⁰ or S;

Y⁵ is (C(R¹⁰)₂)_(q), NR¹⁰(C(R¹⁰)₂)_(q), orNR¹⁰(C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6; and

r is 0, 1, 2, 3, 4, 5, or 6;

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of drug-linker reagents include:

Y⁶ is S, (C(R¹⁰)₂)_(n)S;

Y⁷ is (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

Y⁸ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, or (C(R¹⁰)₂)_(q)S;

Y⁹ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, (C(R¹⁰)₂)_(q)S,OC(O)NR¹⁰(C(R¹⁰)₂)_(q)S, or NR¹⁰C(O)NR¹⁰(C(R¹⁰)₂)_(q)S,

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6;

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of drug-linker reagents include:

Embodiments of drug-linker reagents include:

wherein

Y⁶ is S, (C(R¹⁰)₂)_(n)S;

Y⁷ is (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

Y⁸ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, or (C(R¹⁰)₂)_(q)S;

Y⁹ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, (C(R¹⁰)₂)_(q)S,OC(O)NR¹⁰(C(R¹⁰)₂)_(q)S, or NR¹⁰C(O)NR¹⁰(C(R¹⁰)₂)_(q)S,

Y¹⁰ is OH or N-hydroxysuccinimide;

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of drug-linker reagents include:

Embodiments of drug-linker reagents include:

wherein

Y⁶ is S, (C(R¹⁰)₂)_(n)S;

Y¹¹ is (C(R¹⁰)₂)_(q)O, (C(R¹⁰)₂)_(n)NR¹⁰, (C(R¹⁰)₂)_(q)S,NR¹⁰(C(R¹⁰)₂)_(q)NR¹⁰, NR¹⁰(C(R¹⁰)₂)_(q)O,NR¹⁰(C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q), or O(C(R¹⁰)₂)_(q)O;

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

n is 0, 1, 2, or 3; and

q is 2, 3, 4, 5, or 6.

Linker Reagents

Beta-glucuronide linkers between the antibody and the drug moiety by aresubstrates for cleavage by beta-glucuronidase (Jeffrey et al (2006)Bioconjugate Chem. 17:831-840; WO 2007/011968). The acetal linkage ofbeta-glucuronide releases a phenolic hydroxyl on the aryl ring,potentiating “self-immolation” and 1,6-elimination of thebenzyloxycarbonyl group.

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative spacer has the structure:

where Q is C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, —NO₂ or —CN; and mis an integer ranging from 0-4.

An exemplary phe-lys(Mtr) dipeptide linker reagent having a maleimidestretcher unit and a p-aminobenzyl self-immolative Spacer unit can beprepared according to Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60, and has the structure:

where Mtr is mono-4-methoxytrityl, Q is C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-halogen, —NO₂ or —CN; and m is an integer ranging from 0-4.

The “self-immolative linker”, PABC or PAB (para-aminobenzyloxycarbonyl),attaches the drug moiety to the antibody in the conjugate (Carl et al(1981) J. Med. Chem. 24:479-480; Chakravarty et al (1983) J. Med. Chem.26:638-644; U.S. Pat. No. 6,214,345; US20030130189; US20030096743; U.S.Pat. No. 6,759,509; US20040052793; U.S. Pat. Nos. 6,218,519; 6,835,807;6,268,488; US20040018194; WO98/13059; US20040052793; U.S. Pat. Nos.6,677,435; 5,621,002; US20040121940; WO2004/032828). Other examples ofself-immolative spacers besides PAB include, but are not limited to: (i)aromatic compounds that are electronically similar to the PAB group suchas 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg.Med. Chem. Lett. 9:2237), thiazoles US 2005/0256030), multiple,elongated PAB units (de Groot et al (2001) J. Org. Chem. 66:8815-8830;and ortho or para-aminobenzylacetals; and (ii) homologated styryl PABanalogs (U.S. Pat. No. 7,223,837). Spacers can be used that undergocyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Elimination of amine-containing drugs that aresubstituted at glycine (Kingsbury et al (1984) J. Med. Chem. 27:1447)are also examples of self-immolative spacers useful in ADC.

Linker reagents useful for the antibody drug conjugates of the inventioninclude, but are not limited to: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, 1,8-bis-maleimidodiethyleneglycol(BM(PEO)₂), and 1,11-bis-maleimidotriethyleneglycol (BM(PEO)₃), whichare commercially available from Pierce Biotechnology, Inc.,ThermoScientific, Rockford, Ill., and other reagent suppliers.Bis-maleimide reagents allow the attachment of a free thiol group of acysteine residue of an antibody to a thiol-containing drug moiety,label, or linker intermediate, in a sequential or concurrent fashion.Other functional groups besides maleimide, which are reactive with athiol group of an antibody, nemorubicin metabolite and analog drugmoiety, or linker intermediate include iodoacetamide, bromoacetamide,vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, andisothiocyanate.

Other linker reagents are: N-succinimidyl-4-(2-pyridylthio)pentanoate(SPP), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP, Carlsson etal (1978) Biochem. J. 173:723-737),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Useful linker reagents can also beobtained via other commercial sources, such as Molecular BiosciencesInc. (Boulder, Colo.), or synthesized in accordance with proceduresdescribed in Toki et al (2002) J. Org. Chem. 67:1866-1872; U.S. Pat. No.6,214,345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO03/043583; and WO 04/032828.

The Linker may be a dendritic type linker for covalent attachment ofmore than one drug moiety through a branching, multifunctional linkermoiety to an antibody (US 2006/116422; US 2005/271615; de Groot et al(2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al (2003) Angew.Chem. Int. Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chem. Soc.126:1726-1731; Sun et al (2002) Bioorganic & Medicinal Chemistry Letters12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry11:1761-1768; King et al (2002) Tetrahedron Letters 43:1987-1990).Dendritic linkers can increase the molar ratio of drug to antibody, i.e.loading, which is related to the potency of the ADC. Thus, where anantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic or branched linker.

Antibodies

The antibody unit (Ab) of Formula I includes any unit, type, or class ofantibody that binds or reactively associates or complexes with areceptor, antigen or other receptive moiety associated with a giventarget-cell population. An antibody can be any protein or protein-likemolecule that binds to, complexes with, or reacts with a moiety of acell population sought to be therapeutically or otherwise biologicallymodified. In one aspect, the antibody unit acts to deliver thenemorubicin metabolite and analog drug moiety to the particular targetcell population with which the antibody unit reacts. Such antibodiesinclude, but are not limited to, large molecular weight proteins suchas, full-length antibodies and antibody fragments. The antibodies ofFormula I allow attaining high concentrations of active metabolitemolecules in cancer cells. Intracellular targeting may be achieved bymethods and compounds which allow accumulation or retention ofbiologically active agents inside cells. Such effective targeting may beapplicable to a variety of therapeutic formulations and procedures.

In one embodiment, the ADC specifically binds to a receptor encoded byan ErbB gene, such as EGFR, HER2, HER3 and HER4. The ADC mayspecifically bind to the extracellular domain of the HER2 receptor. TheADC may inhibit growth of tumor cells which overexpress HER2 receptor.

In another embodiment, the antibody (Ab) of Formula I is a humanizedantibody such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4,huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab).

The antibodies of the invention include cysteine-engineered antibodieswhere one or more amino acids of any form of wild-type or parentantibody is replaced with a cysteine amino acid. The engineered cysteineamino acid is a free cysteine acid and not part of an intrachain orinterchain disulfide unit. Any form, type, or variant of antibody may beso engineered, i.e. mutated. For example, a parent Fab antibody fragmentmay be engineered to form a cysteine engineered Fab, referred to hereinas “ThioFab.” Similarly, a parent monoclonal antibody may be engineeredto form a “ThioMab.” It should be noted that a single site mutationyields a single engineered cysteine residue in a ThioFab, while a singlesite mutation yields two engineered cysteine residues in a ThioMab, dueto the dimeric nature of the IgG antibody. The cysteine engineeredantibodies of the invention include monoclonal antibodies, humanized orchimeric monoclonal antibodies, antigen-binding fragments of antibodies,fusion polypeptides and analogs that preferentially bind cell-associatedpolypeptides.

Cysteine-engineered antibodies have been designed as Fab antibodyfragments (thioFab) and expressed as full-length, IgG monoclonal(thioMab) antibodies (US 2007/0092940, the contents of which areincorporated by reference). ThioFab and ThioMab antibodies have beenconjugated through linkers at the newly introduced cysteine thiols withthiol-reactive linker reagents and drug-linker reagents to prepareantibody drug conjugates (Thio ADC).

Antibodies comprising the antibody-drug conjugates of the inventionpreferably retain the antigen binding capability of their native, wildtype counterparts. Thus, antibodies of the invention are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, cell survival regulatoryfactors, cell proliferation regulatory factors, molecules associatedwith (for e.g., known or suspected to contribute functionally to) tissuedevelopment or differentiation, lymphokines, cytokines, moleculesinvolved in cell cycle regulation, molecules involved in vasculogenesisand molecules associated with (for e.g., known or suspected tocontribute functionally to) angiogenesis. The tumor-associated antigenmay be a cluster differentiation factor (i.e., a CD protein). An antigento which an antibody of the invention is capable of binding may be amember of a subset of one of the above-mentioned categories, wherein theother subset(s) of said category comprise other molecules/antigens thathave a distinct characteristic (with respect to the antigen ofinterest).

In one embodiment, the antibody of the antibody-drug conjugates (ADC)specifically binds to a receptor encoded by an ErbB gene. The antibodymay bind specifically to an ErbB receptor selected from EGFR, HER2, HER3and HER4. The ADC may specifically bind to the extracellular domain(ECD) of the HER2 receptor and inhibit the growth of tumor cells whichoverexpress HER2 receptor. The antibody of the ADC may be a monoclonalantibody, e.g. a murine monoclonal antibody, a chimeric antibody, or ahumanized antibody. A humanized antibody may be huMAb4D5-1, huMAb4D5-2,huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8(trastuzumab). The antibody may be an antibody fragment, e.g. a Fabfragment.

Antibodies in Formula I antibody-drug conjugates (ADC) and which may beuseful in the treatment of cancer include, but are not limited to,antibodies against cell surface receptors and tumor-associated antigens(TAA). Such tumor-associated antigens are known in the art, and canprepared for use in generating antibodies using methods and informationwhich are well known in the art. In attempts to discover effectivecellular targets for cancer diagnosis and therapy, researchers havesought to identify transmembrane or otherwise tumor-associatedpolypeptides that are specifically expressed on the surface of one ormore particular type(s) of cancer cell as compared to on one or morenormal non-cancerous cell(s). Often, such tumor-associated polypeptidesare more abundantly expressed on the surface of the cancer cells ascompared to on the surface of the non-cancerous cells. Theidentification of such tumor-associated cell surface antigenpolypeptides has given rise to the ability to specifically target cancercells for destruction via antibody-based therapies.

Examples of TAA include, but are not limited to, Tumor-AssociatedAntigens (1)-(36) listed below. For convenience, information relating tothese antigens, all of which are known in the art, is listed below andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(36) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

Tumor-Associated Antigens (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_001203); ten Dijke, P., et al Science 264(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004063362(Claim 2); WO2003042661 (Claim 12); US2003134790-A1 (Page 38-39);WO2002102235 (Claim 13; Page 296); WO2003055443 (Page 91-92);WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim 6);WO2003024392 (Claim 2; FIG. 112); WO200298358 (Claim 1; Page 183);WO200254940 (Page 100-101); WO200259377(Page 349-350); WO200230268(Claim 27; Page 376); WO200148204 (Example; FIG. 4); NP_001194 bonemorphogenetic protein receptor, type IB/pid=NP_001194.1;Cross-references: MIM:603248; NP_001194.1; AY065994

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486); Biochem.Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291(1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33;Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (Claim 12; Page 150); US 20050107595; US 20050106644;NP_003477 solute carrier family 7 (cationic amino acid transporter, y+system), member 5/pid=NP_003477.3—Homo sapiens; Cross-references:MIM:600182; NP_003477.3; NM_015923; NM_003486_1

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_012449); Cancer Res. 61 (15), 5857-5860 (2001), Hubert,R. S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528);WO2004065577 (Claim 6); WO2004027049 (FIG. 1L); EP1394274 (Example 11);WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example5); US2003185830 (Example 5); US2003064397 (FIG. 2); WO200289747(Example 5; Page 618-619); WO2003022995 (Example 9; FIG. 13A, Example53; Page 173, Example 2; FIG. 2A); NP_036581 six transmembraneepithelial antigen of the prostate; Cross-references: MIM:604415;NP_036581.1; NM_012449_1

(4) 0772P(CA125, MUC16, Genbank accession no. AF361486); J. Biol. Chem.276 (29):27371-27375 (2001)); WO2004045553 (Claim 14); WO200292836(Claim 6; FIG. 12); WO200283866 (Claim 15; Page 116-121); US2003124140(Example 16); US2003091580 (Claim 6); WO200206317 (Claim 6; Page400-408); Cross-references: GI:34501467; AAK74120.3; AF361486_1

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_005823); Yamaguchi, N., et al Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol.Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim 14);(WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_005814.2; NM_005823_1

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_006424); J. Biol. Chem. 277 (22):19665-19672(2002), Genomics 62 (2):281-284 (1999), Feild, J. A., et al (1999)Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (Claim 2);EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569(Claim 1; Page 17-19); WO200157188 (Claim 20; Page 329); WO2004032842(Example IV); WO200175177 (Claim 24; Page 139-140); Cross-references:MIM:604217; NP_006415.1; NM_006424_1

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878); Nagase T., et al(2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1); WO2003003984(Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page41-43, 48-58); WO2003054152 (Claim 20); WO2003101400 (Claim 11);Accession: □9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737;

(8) PSCA hlg (2700050C12R1k, C530008O16R1k, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY³⁵⁸⁶²⁸); Ross et al (2002)Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180 (Claim12); US2004044179 (Claim 11); US2003096961 (Claim 11); US2003232056(Example 5); WO2003105758 (Claim 12); US2003206918 (Example 5);EP1347046 (Claim 1); WO2003025148 (Claim 20); Cross-references:GI:37182378; AAQ88991.1; AY358628_1

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004045516 (Claim 1); WO2004048938 (Example2); WO2004040000 (Claim 151); WO2003087768 (Claim 1); WO2003016475(Claim 1); WO2003016475 (Claim 1); WO200261087 (FIG. 1); WO2003016494(FIG. 6); WO2003025138 (Claim 12; Page 144); WO200198351 (Claim 1; Page124-125); EP522868 (Claim 8; FIG. 2); WO200177172 (Claim 1; Page297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No.5,773,223 (Claim 1a; Col 31-34); WO2004001004.

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM_017763); WO2003104275 (Claim 1); WO2004046342 (Example 2);WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621(Claim 1); WO2003024392 (Claim 2; FIG. 93); WO200166689 (Example 6);Cross-references: LocusID:54894; NP_060233.2; NM_017763_1

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP 1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138); Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (Claim 1; FIG. 1);WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; FIG. 4B);WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim22); WO2003042661 (Claim 12); US2003060612 (Claim 12; FIG. 10);WO200226822 (Claim 23; FIG. 2); WO200216429 (Claim 12; FIG. 10);Cross-references: GI:22655488; AAN04080.1; AF455138_1

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_017636); Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98(19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); US2003143557 (Claim 4); WO200040614 (Claim 14;Page 100-103); WO200210382 (Claim 1; FIG. 9A); WO2003042661 (Claim 12);WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794(Claim 14; FIG. 1A-D); Cross-references: MIM:606936; NP_060106.2;NM_017636_1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_003203 or NM_003212);Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); WO2003083041(Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53);WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105);WO200222808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2); Cross-references: MIM:187395;NP_003203.1; NM_003212_1

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004); Fujisaku et al(1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al J. Exp. Med.167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad. Sci. U.S.A. 84,9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; WeisJ. J., et al Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S.K., et al (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4);US2004005538 (Example 1); WO2003062401 (Claim 9); WO2004045520 (Example4); WO9102536 (FIG. 9.1-9.9); WO2004020595 (Claim 1); Accession: P20023;Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_000626 or 11038674); Proc. Natl. Acad. Sci.U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Mulleret al (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (claim 2,FIG. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401(claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15);U.S. Pat. No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B); WO200055351(claim 11, pages 1145-1146); Cross-references: MIM: 147245; NP 000617.1;NM 000626_1

(16) FcRH2 (IFGP4, IRTA4, SPAPlA (SH2 domain containing phosphataseanchor protein 1a), SPAPIB, SPAPIC, Genbank accession no. NM_030764,AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (Claim 2);WO2003077836; WO200138490 (Claim 5; FIG. 18D-1-18D-2); WO2003097803(Claim 12); WO2003089624 (Claim 25); Cross-references: MIM:606509;NP_110391.2; NM_030764_1

(17) HER2 (ErbB2, Genbank accession no. M11730); Coussens L., et alScience (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319,230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82,6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165, 869-880, 2004;Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., etal Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622;WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1);US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG.1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession:P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et alGenomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res.Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc. Natl. Acad. Sci.U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393 (Claim 7);WO2004044178 (Example 4); WO2004031238; WO2003042661 (Claim 12);WO200278524 (Example 2); WO200286443 (Claim 27; Page 427); WO200260317(Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL;M18728

(19) MDP (DPEP1, Genbank accession no. BC017023); Proc. Natl. Acad. Sci.U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (Claim 1); WO200264798(Claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9);Cross-references: MIM:179780; AAH17023.1; BC017023_1

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971); Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al Nature425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001; DumoutierL., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al J.Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003)Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59); Accession:Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053); Gary S. C.,et al Gene 256, 139-147, 2000; Clark H. F., et al Genome Res. 13,2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A.99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373 (Claim 11);US2003119131 (Claim 1; FIG. 52); US2003119122 (Claim 1; FIG. 52);US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52); US2003119129(Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1);

(22) EphB2R (DRT, ERK, HekS, EPHT3, Tyro5, Genbank accession no.NM_004442); Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991)Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998),Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12);WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583(Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42);Cross-references: MIM:600997; NP_004433.2; NM_004442_1

(23) ASLG659 (B7h, Genbank accession no. AX092328); US20040101899 (Claim2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3); US2003165504 (Claim1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235(Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6;FIG. 10); WO200194641 (Claim 12; FIG. 7b); WO200202624 (Claim 13; FIG.1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2;Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469);WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192);WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12);WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318;

(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436); Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1);WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);WO200032752 (Claim 18; FIG. 1); WO9851805 (Claim 17; Page 97); WO9851824(Claim 10; Page 94); WO9840403 (Claim 2; FIG. 1B); Accession: 043653;EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGICfusion-partner-like protein/pid=AAP14954.1—Homo sapiens (human);WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example3, Claim 20); US2003194704 (Claim 45); Cross-references: GI:30102449;AAP14954.1; AY260763_1

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1—Homosapiens; Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); WO200224909(Example 3; FIG. 3); Cross-references: MIM:606269; NP_443177.1;NM_052945_1; AF132600

(27) CD22 (B-cell receptor CD22-β isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al (1991)J. Exp. Med. 173:137-146; WO2003072036 (Claim 1; FIG. 1);Cross-references: MIM:107266; NP_001762.1; NM_001771_1

(28) CD79a (CD79A, CD79a, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation); 226 aa), pI: 4.84, MW: 25028 TM: 2[P] Gene Chromosome: 19q13.2, Genbank accession No. NP_001774.10);WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S.Pat. No. 5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531;Mueller et al (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al(1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa),pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accessionNo. NP_001707.1); WO2004040000; WO2004015426; US2003105292 (Example 2);U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188(Claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1,pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38);U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);WO9217497 (claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol.22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI:6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_002111.1); Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson etal (1989) Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci. USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.NP_002552.2); Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004047749;WO2003072035 (claim 10); Touchman et al (2000) Genome Res. 10:165-173;WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1);WO2003029277 (page 82)

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa), pI:8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No.NP_001773.1); WO2004042346 (claim 65); WO2003026493 (pages 51-52,57-58); WO200075655 (pages 105-106); Von Hoegen et al (1990) J. Immunol.144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci. USA99:16899-16903

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa), pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_005573.1); US2002193567; WO9707198 (claim 11, pages 39-42); Miura etal (1996) Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822;WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages24-26)

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa), pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_443170.1); WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2);Davis et al (2001) Proc. Natl. Acad. Sci. USA 98(17):9772-9777;WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7)

(35) IRTA2 (FcRH5, Immunoglobulin superfamily receptor translocationassociated 2, a putative immunoreceptor with possible roles in B celldevelopment and lymphomagenesis; deregulation of the gene bytranslocation occurs in some B cell malignancies); 977 aa), pI: 6.88 MW:106468 TM: 1 [P] Gene Chromosome: 1q21, (Genbank accession No. Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423,AK090475, AL834187, AY358085; Mouse: AK089756, AY158090, AY506558;NP_112571.1); WO2003024392 (claim 2, FIG. 97); Nakayama et al (2000)Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836; WO200138490(claim 3, FIG. 18B-1-18B-2)

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;(Genbank accession No. AF179274; AY358907, CAF85723, CQ782436);WO2004074320; JP2004113151; WO2003042661; WO2003009814; EP1295944 (pages69-70); WO200230268 (page 329); WO200190304; US2004249130; US2004022727;WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579;U.S. Pat. Nos. 6,410,506; 6,642,006; Horie et al (2000) Genomics67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun.266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones etal (2001) Int J Cancer. October 15; 94(2):178-84.

Antibody-Drug Conjugates

The compounds of the invention include those with utility for anticanceractivity. In particular, the compounds include an antibody conjugated,i.e. covalently attached by a linker, to a nemorubicin metabolite andanalog drug moiety where the drug when not conjugated to an antibody hasa cytotoxic or cytostatic effect. The biological activity of the drugmoiety is thus modulated by conjugation to an antibody. Theantibody-drug conjugates (ADC) of the invention may selectively deliveran effective dose of a cytotoxic agent to tumor tissue whereby greaterselectivity, i.e. a lower efficacious dose may be achieved.

In one embodiment, the bioavailability of the ADC, or an intracellularmetabolite of the ADC, is improved in a mammal when compared to thecorresponding nemorubicin metabolite and analog compound alone. Also,the bioavailability of the ADC, or an intracellular metabolite of theADC is improved in a mammal when compared to the corresponding antibodyalone (antibody of the ADC, without the drug moiety or linker).

In one embodiment, the nemorubicin metabolite and analog drug moiety ofthe ADC is not cleaved from the antibody until the antibody-drugconjugate binds to a cell-surface receptor or enters a cell with acell-surface receptor specific for the antibody of the antibody-drugconjugate. The drug moiety may be cleaved from the antibody after theantibody-drug conjugate enters the cell. The nemorubicin metabolite andanalog drug moiety may be intracellularly cleaved in a mammal from theantibody of the compound, or an intracellular metabolite of thecompound, by enzymatic action, hydrolysis, oxidation, or othermechanism.

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic subsituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, G. T. (1996) BioconjugateTechniques; Academic Press: New York, p 234-242). In another embodiment,proteins containing N-terminal serine or threonine residues can reactwith sodium meta-periodate, resulting in production of an aldehyde inplace of the first amino acid (Geoghegan & Stroh, (1992) BioconjugateChem. 3:138-146; U.S. Pat. No. 5,362,852). Such aldehyde can be reactedwith a drug moiety or linker nucleophile.

Antibody-drug conjugates (ADC) may be represented by Formula I:Ab-(L-D)_(p)  I

comprising an antibody covalently attached by a linker to one or morenemorubicin metabolite or analog drug moieties, or a pharmaceuticallyacceptable salt thereof, wherein:

Ab is an antibody;

L is a linker; and

D is a nemorubicin metabolite or analog drug moiety having thestructure:

wherein:

Y is N—X⁶ or O;

L is attached at one of X¹, X², X³, X⁴, X⁵, or X⁶; and

p is 1, 2, 3, 4, 5, 6, 7, or 8.

The drug to antibody ratio or drug loading is represented by p forFormula I compounds. The drug loading value p is 1 to 8. Formula Icompounds include all mixtures of variously loaded and attachedantibody-drug conjugates where 1, 2, 3, 4, 5, 6, 7, and 8 drug moietiesare covalently attached to the antibody.

Embodiments of antibody-drug conjugates include:

where A_(a) is a divalent unit, such as MC (maleimidocaproyl), MP(maleimidopropanoyl) or MPEG(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)acetyl),capable of linking an antibody (Ab) to an amino acid unit, such asvaline-citrulline; and Y_(y) is a divalent unit, such as PAB(para-aminobenzyloxycarbonyl) which links an amino acid unit to the drugmoiety (D) when an amino acid unit is present. In other embodiments,A_(a) links Y_(y) directly to the drug moiety when the amino acid unitis absent. In other embodiments, the Y_(y) unit links directly the drugmoiety to the antibody unit when both the amino acid unit and the A_(a)unit are absent.

Exemplary antibody-disulfide linker drug conjugates are represented bythe structures:

The disulfide linker SPP may be constructed with linker reagentN-succinimidyl 4-(2-pyridylthio) pentanoate.

Embodiments of antibody-drug conjugates include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

R¹ and R² are independently an amino acid side chain selected fromhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the following structures:

R³ is H, C₁-C₈ alkyl, NR¹⁰C(O)R¹⁰, or C(O)CH₃;

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃; and

n is 0, 1, 2, 3, 4, 5, or 6.

Embodiments of antibody-drug conjugates include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, O—(C₁-C₆ alkyl)-NR¹⁰, O(C₁-C₆ alkyl)O, orOC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰;

R¹ and R² are independently an amino acid side chain selected fromhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the structures:

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

n is independently 0, 1, 2, 3, 4, 5, or 6.

Embodiments of antibody-drug conjugates include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, OC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰; and

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃.

Embodiments of antibody-drug conjugates include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, OC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰;

Y³ is (C(R¹⁰)₂)_(r);

R¹ and R² are independently an amino acid side chain selected fromhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl, and the structures:

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6;

r is 0, 1, 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of antibody-drug conjugates include:

Embodiments of antibody-drug conjugates include:

wherein

Y¹ is C(O)(C(R¹⁰)₂)_(q), (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q)

q is 2, 3, 4, 5, or 6.

Y² is O, NR¹⁰, S, OC(O)NR¹⁰—(C₁-C₆ alkyl)-NR¹⁰;

Y³ is (C(R¹⁰)₂)_(r);

R¹ is independently an amino acid side chain selected from hydrogen,methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, and thestructures:

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6;

r is 0, 1, 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of antibody-drug conjugates include:

wherein

Y³ is (C(R¹⁰)₂)_(r);

Y⁴ is O, NR¹⁰ or S;

Y⁵ is (C(R¹⁰)₂)_(q), NR¹⁰(C(R¹⁰)₂)_(q), orNR¹⁰(C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6; and

r is 0, 1, 2, 3, 4, 5, or 6;

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of antibody-drug conjugates include:

Y⁶ is S, (C(R¹⁰)₂)_(n)S;

Y⁷ is (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

Y⁸ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, or (C(R¹⁰)₂)_(q)S;

Y⁹ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, (C(R¹⁰)₂)_(q)S,OC(O)NR¹⁰(C(R¹⁰)₂)_(q)S, or NR¹⁰C(O)NR¹⁰(C(R¹⁰)₂)_(q)S,

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6;

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of antibody-drug conjugates include:

Embodiments of antibody-drug conjugates comprise SMCC linkers and thenemorubicin metabolite or analog drug moiety, represented as Ab-MCC-D:

wherein

Y⁶ is S, (C(R¹⁰)₂)_(n)S;

Y⁷ is (C(R¹⁰)₂)_(q), or (C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q);

Y⁸ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, or (C(R¹⁰)₂)_(q)S;

Y⁹ is S, NR¹⁰(C(R¹⁰)₂)_(q)S, O(C(R¹⁰)₂)_(q)S, (C(R¹⁰)₂)_(q)S,OC(O)NR(C(R¹⁰)₂)_(q)S, or NR¹⁰C(O)NR¹⁰(C(R¹⁰)₂)_(q)S,

Y¹⁰ is OH or N-hydroxysuccinimide;

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

q is 2, 3, 4, 5, or 6; and

n is 1, 2, 3, 4, 5, 6, or 7.

Embodiments of antibody-drug conjugates include:

Embodiments of antibody-drug conjugates include:

wherein

Y⁶ is S, (C(R¹⁰)₂)_(n)S;

Y¹¹ is (C(R¹⁰)₂)_(q)O, (C(R¹⁰)₂)_(n)NR¹⁰, (C(R¹⁰)₂)_(q)S,NR¹⁰(C(R¹⁰)₂)_(q)NR NR¹⁰(C(R¹⁰)₂)_(q)O, NR¹⁰(C(R¹⁰)₂)_(q)O(C(R¹⁰)₂)_(q),or O(C(R¹⁰)₂)_(q)O;

each R¹⁰ is independently selected from H, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl, optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₂OH, —CH₂C₆H₅, —CN, —CF₃,—CO₂H, —CONH₂, —CONHCH₃, —NO₂, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —OH,—OCH₃, —OCH₂CH₃, —S(O)₂NH₂, and —S(O)₂CH₃;

n is 0, 1, 2, or 3; and

q is 2, 3, 4, 5, or 6.

Drug Loading

The drug loading is represented by p in an antibody-drug conjugatemolecule of Formula I, the average number of nemorubicin metabolite andanalog drugs per antibody. Drug loading may range from 1 to 8 drugs (D)per antibody (Ab), i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moietiesare covalently attached to the antibody. Compositions of ADC of FormulaI include collections of antibodies conjugated with a range of drugs,from 1 to 8. The average number of drugs per antibody in preparations ofADC from conjugation reactions may be characterized by conventionalmeans such as mass spectroscopy, ELISA assay, electrophoresis, and HPLC.The quantitative distribution of ADC in terms of p may also bedetermined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblett et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p>5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thedrug-linker intermediate (D-L) or linker reagent. Only the most reactivelysine groups may react with an amine-reactive linker reagent. Also,only the most reactive cysteine thiol groups may react with athiol-reactive linker reagent. Generally, antibodies do not containmany, if any, free and reactive cysteine thiol groups which may belinked to a drug moiety. Most cysteine thiol residues in the antibodiesof the compounds exist as disulfide bridges and must be reduced with areducing agent such as dithiothreitol (DTT) or TCEP, under partial ortotal reducing conditions. Additionally, the antibody must be subjectedto denaturing conditions to reveal reactive nucleophilic groups such aslysine or cysteine. The loading (drug/antibody ratio) of an ADC may becontrolled in several different manners, including: (i) limiting themolar excess of drug-linker intermediate (D-L) or linker reagentrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(US 2007/0092940). The engineered cysteine thiols may react with linkerreagents or the drug-linker reagents of the present invention which havethiol-reactive, electrophilic groups such as maleimide or alpha-haloamides to form ADC with cysteine engineered antibodies and thenemorubicin metabolite or analog drug moieties. The location of the drugmoiety can thus be designed, controlled, and known. The drug loading canbe controlled since the engineered cysteine thiol groups typically reactwith thiol-reactive linker reagents or drug-linker reagents in highyield. Engineering an IgG antibody to introduce a cysteine amino acid bysubstitution at a single site on the heavy or light chain gives two newcysteines on the symmetrical antibody. A drug loading near 2 can beachieved and near homogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Preparation of Antibody-Drug Conjugates

The ADC of Formula I may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a nucleophilic group oran electrophilic group of an antibody with a bivalent linker reagent, toform antibody-linker intermediate Ab-L, via a covalent bond, followed byreaction with an activated drug moiety reagent; and (2) reaction of anucleophilic group or an electrophilic group of a drug moiety reagentwith a linker reagent, to form drug-linker reagent D-L, via a covalentbond, followed by reaction with the nucleophilic group or anelectrophilic group of an antibody. Conjugation methods (1) and (2) maybe employed with a variety of antibodies, drug moieties, and linkers toprepare the antibody-drug conjugates of Formula I.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). Each cysteinedisulfide bridge will thus form, theoretically, two reactive thiolnucleophiles. Additional nucleophilic groups can be introduced intoantibodies through the reaction of lysines with 2-iminothiolane (Traut'sreagent) resulting in conversion of an amine into a thiol.

Antibody-drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, G. T. (1996) BioconjugateTechniques; Academic Press: New York, p 234-242). In another embodiment,proteins containing N-terminal serine or threonine residues can reactwith sodium meta-periodate, resulting in production of an aldehyde inplace of the first amino acid (Geoghegan & Stroh, (1992) BioconjugateChem. 3:138-146; U.S. Pat. No. 5,362,852). Such aldehyde can be reactedwith a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups. Reactive nucleophilic groups may beintroduced on the nemorubicin metabolite and analog compounds bystandard functional group intercoversions. For example, hydroxyl groupsmay be converted to thiol groups by Mitsunobu-type reactions, to formthiol-modified drug compounds.

Screening for Antibody-Drug Conjugates (ADC) Directed AgainstTumor-Assoclated Antigens and Cell Surface Receptors

Assay methods for detecting cancer cells comprise exposing cells to anantibody-drug conjugate compound, and determining the extent of bindingof the antibody-drug conjugate compound to the cells. Formula I ADCcompounds which are identified in the animal models and cell-basedassays can be further tested in tumor-bearing higher primates and humanclinical trials.

Transgenic animals and cell lines are particularly useful in screeningantibody-drug conjugates (ADC) that have potential as prophylactic ortherapeutic treatments of diseases or disorders involving overexpressionof tumor-associated antigens and cell surface receptors, e.g. HER2 (U.S.Pat. No. 6,632,979). Screening for a useful ADC may involveadministering candidate ADC over a range of doses to the transgenicanimal, and assaying at various time points for the effect(s) of the ADCon the disease or disorder being evaluated. Alternatively, oradditionally, the drug can be administered prior to or simultaneouslywith exposure to an inducer of the disease, if applicable. Candidate ADCmay be screened serially and individually, or in parallel under mediumor high-throughput screening format. The rate at which ADC may bescreened for utility for prophylactic or therapeutic treatments ofdiseases or disorders is limited only by the rate of synthesis orscreening methodology, including detecting/measuring/analysis of data.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable breast cancer cell line into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the formation oftumors from the transplanted cell line. The invention also concerns amethod of screening ADC candidates for the treatment of a disease ordisorder characterized by the overexpression of a receptor proteincomprising (a) contacting cells from a stable breast cancer cell linewith a drug candidate and (b) evaluating the ability of the ADCcandidate to inhibit the growth of the stable cell line.

One embodiment is a screening method comprising (a) contacting cellsfrom a stable breast cancer cell line with an ADC drug candidate and (b)evaluating the ability of the ADC candidate to induce cell death, induceapoptosis, block heregulin binding, block ligand-stimulated tyrosinephosphorylation, or block ligand activation of HER2. Another embodimentthe ability of the ADC candidate to is evaluated. In another embodimentthe ability of the ADC candidate to is evaluated.

Another embodiment is a screening method comprising (a) administering anADC drug candidate to a transgenic non-human mammal that overexpresses,e.g. in its mammary gland cells, a native human protein, e.g. HER2 or afragment thereof, wherein such transgenic mammal has stably integratedinto its genome a nucleic acid sequence encoding the native humanprotein or a fragment thereof having the biological activity of thenative human protein, operably linked to transcriptional regulatorysequences directing its expression, and develops a tumor. Candidate ADCare screened by being administered to the transgenic animal over a rangeof doses, and evaluating the animal's physiological response to thecompounds over time. Administration may be oral, or by suitableinjection, depending on the chemical nature of the compound beingevaluated. In some cases, it may be appropriate to administer thecompound in conjunction with co-factors that would enhance the efficacyof the compound. If cell lines derived from the subject transgenicanimals are used to screen for compounds useful in treating variousdisorders associated with overexpression of certain tumor-associatedantigen proteins or cell surface receptors, e.g. HER2-overexpression. Toidentify growth inhibitory ADC compounds that specifically target HER2,one may screen for ADC which inhibit the growth of HER2-overexpressingcancer cells derived from transgenic animals (U.S. Pat. No. 5,677,171).

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells havingtumor-associated antigens or receptor proteins to the antibody of theADC in a cell culture medium; culturing the cells for a period fromabout 6 hours to about 5 days; and measuring cell viability. Cell-basedin vitro assays may be used to measure viability, i.e. proliferation(IC₅₀), cytotoxicity (EC₅₀), and induction of apoptosis (caspaseactivation) of the ADC. The CellTiter-Glo® Luminescent Cell ViabilityAssay is a commercially available (Promega Corp., Madison, Wis.),homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713; 5,700,670).This cell proliferation assay determines the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay isconducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

In Vivo Serum Clearance and Stability in Mice

Serum clearance and stability of ADC may be investigated in nude, naive(without tumors received by exogenous grafts) mice. A difference in theamount of total antibody and ADC indicates cleavage of the linker andseparation of the antibody from its drug moiety.

In Vivo Efficacy

The efficacy of the antibody-drug conjugates of the invention may bemeasured in vivo by implanting allografts or xenografts of cancer cellsor primary tumors in rodents and treating the tumors with ADC. Variableresults are to be expected depending on the cell line, the specificityof antibody binding of the ADC to receptors present on the cancer cells,dosing regimen, and other factors. For example, the in vivo efficacy ofanti-HER2 ADC may be measured by a high expressing HER2 transgenicexplant mouse model. An allograft may be propagated from the Fo5 mmtvtransgenic mouse which does not respond to, or responds poorly to,HERCEPTIN® therapy. Subjects are treated once with ADC and monitoredover 3-6 weeks to measure the time to tumor doubling, log cell kill, andtumor shrinkage. Follow up dose-response and multi-dose experiments mayfurther be conducted.

Rodent Toxicity

Antibody-drug conjugates and an ADC-minus control, “Vehicle”, may beevaluated in an acute toxicity rat model (Brown et al (2002) CancerChemother. Pharmacol. 50:333-340). Toxicity of ADC are investigated bytreatment of female Sprague-Dawley rats with the ADC and subsequentinspection and analysis of the effects on various organs. Based on grossobservations (body weights), clinical pathology parameters (serumchemistry and hematology) and histopathology, the toxicity of ADC may beobserved, characterized, and measured.

A multi-day acute toxicity study in adolescent female rats may beconducted by one or more doses of a candidate ADC, a control ADC, freenemorubicin metabolite and analog compound and a control Vehicle (day0). Body weight is measured periodically. Clinical chemistry, serumenzymes and hematology analysis is also conducted periodically;concluding with complete necropsy with histopathological assessment.Toxicity signals included the clinical observation of weight loss,considering that weight loss, or weight change relative to animals dosedonly with Vehicle in animals after dosing with ADC, is a gross andgeneral indicator of systemic or localized toxicity. Hepatotoxicity maybe measured by: (i) elevated liver enzymes such as AST (aspartateaminotransferase), ALT (alanine aminotransferase), GGT (g-glutamyltransferase); (ii) increased numbers of mitotic and apoptotic figures;and (iii) hepatocyte necrosis. Hematolymphoid toxicity is observed bydepletion of leukocytes, primarily granuloctyes (neutrophils), and/orplatelets, and lymphoid organ involvement, i.e. atrophy or apoptoticactivity. Toxicity is also noted by gastrointestinal tract lesions suchas increased numbers of mitotic and apoptotic figures and degenerativeentercolitis.

Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

Therapeutic antibody-drug conjugates (ADC) may be administered by anyroute appropriate to the condition to be treated. The ADC will typicallybe administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal, bolus, intratumorinjection or epidural (Shire et al (2004) J. Pharm. Sciences93(6):1390-1402). Pharmaceutical formulations of therapeuticantibody-drug conjugates (ADC) are typically prepared for parenteraladministration with a pharmaceutically acceptable parenteral vehicle andin a unit dosage injectable form. An antibody-drug conjugate (ADC)having the desired degree of purity is optionally mixed withpharmaceutically acceptable diluents, carriers, excipients orstabilizers, in the form of a lyophilized formulation or an aqueoussolution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol,A. Ed.).

Acceptable parenteral vehicles, diluents, carriers, excipients, andstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include: (i) buffers such as phosphate, citrate, dibasiccalcium phosphate, magnesium stearate, and other organic acids; (ii)antioxidants including ascorbic acid and methionine; (iii) preservatives(such as octadecyldimethylbenzyl ammonium chloride; hexamethoniumchloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; (iv) alkyl parabens such as methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); (v) lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; (vi) hydrophilicpolymers such as polyvinylpyrrolidone; (vii) amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine; (viii)monosaccharides, disaccharides, and other carbohydrates includingglucose, lactose, sucrose, mannitol, trehalose, sodium starch glycolate,sorbitol mannose, carboxymethylcellulose, or dextrins; (ix) chelatingagents such as EDTA; (x) salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); (xi) non-ionic surfactants suchas TWEEN™, PLURONICS™ or polyethylene glycol (PEG); (xii) glidants orgranulating agents such as magnesium stearate, carboxymethylcellulose,talc, silica, and hydrogenated vegetable oil; (xiii) disintegrant suchas crosprovidone, sodium starch glycolate or cornstarch; (xiv)thickening agents such as gelatin and polyethylene glycol; (xv) entericcoatings such as triethyl citrate; and/or (xvi) taste or texturemodifiers, antifoaming agents, pigments, and dessicants. For example,lyophilized anti-ErbB2 antibody formulations are described in WO97/04801, expressly incorporated herein by reference. An exemplaryformulation of an ADC contains about 100 mg/ml of trehalose(2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5-triol;C₁₂H₂₂O₁₁; CAS Number 99-20-7) and about 0.1% TWEEN™ 20 (polysorbate 20;dodecanoic acid2-[2-[3,4-bis(2-hydroxyethoxy)tetrahydrofuran-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethylester; C₂₆H₅₀O₁₀; CAS Number 9005-64-5) at approximately pH 6.

Pharmaceutical formulations of a therapeutic antibody-drug conjugate(ADC) may contain certain amounts of unreacted drug moiety (D),antibody-linker intermediate (Ab-L), and/or drug-linker intermediate(D-L), as a consequence of incomplete purification and separation ofexcess reagents, impurities, and by-products, in the process of makingthe ADC; or time/temperature hydrolysis or degradation upon storage ofthe bulk ADC or formulated ADC composition. For example, a formulationof the ADC may contain a detectable amount of free drug. Alternatively,or in addition to, it may contain a detectable amount of drug-linkerintermediate. Alternatively, or in addition to, it may contain adetectable amount of the antibody. The active pharmaceutical ingredientsmay also be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi permeable matrices of solidhydrophobic polymers containing the ADC, which matrices are in the formof shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

Formulations may conveniently be presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy.Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared under sterile conditions and byuniformly and intimately bringing into association the ADC with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Aqueous suspensions contain the active materials (ADC) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

The pharmaceutical compositions of ADC may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to 500 μg of the active ingredient per milliliter of solution inorder that infusion of a suitable volume at a rate of about 30 mL/hr canoccur. Subcutaneous (bolus) administration may be effected with about1.5 ml or less of total volume and a concentration of about 100 mg ADCper ml. For ADC that require frequent and chronic administration, thesubcutaneous route may be employed, such as by pre-filled syringe orautoinjector device technology.

As a general proposition, the initial pharmaceutically effective amountof ADC administered per dose will be in the range of about 0.01-100mg/kg, namely about 0.1 to 20 mg/kg of patient body weight per day, withthe typical initial range of compound used being 0.3 to 15 mg/kg/day.For example, human patients may be initially dosed at about 1.5 mg ADCper kg patient body weight. The dose may be escalated to the maximallytolerated dose (MTD). The dosing schedule may be about every 3 weeks,but according to diagnosed condition or response, the schedule may bemore or less frequent. The dose may be further adjusted during thecourse of treatment to be at or below MTD which can be safelyadministered for multiple cycles, such as about 4 or more.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Although oral administration of protein therapeutics are generallydisfavored due to poor bioavailability due to limited absorption,hydrolysis or denaturation in the gut, formulations of ADC suitable fororal administration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the ADC.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Exemplary unit dosage formulations contain a dailydose or unit daily sub-dose, or an appropriate fraction thereof, of theactive ingredient.

Antibody-Drug Conjugate Treatments

Formula I ADC may be used to treat various diseases or disorders in apatient, such as cancer and autoimmune conditions including thosecharacterized by the overexpression of a tumor-associated antigen.Exemplary conditions or disorders include benign or malignant tumors;leukemia and lymphoid malignancies; other disorders such as neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologicdisorders. Cancer types susceptible to ADC treatment include those whichare characterized by the overexpression of certain tumor associatedantigens or cell surface receptors, e.g. HER2.

One method is for the treatment of cancer in a mammal, wherein thecancer is characterized by the overexpression of an ErbB receptor. Themammal optionally does not respond, or responds poorly, to treatmentwith an unconjugated anti-ErbB antibody. The method comprisesadministering to the mammal a therapeutically effective amount of anantibody-drug conjugate compound. The growth of tumor cells thatoverexpress a growth factor receptor such as HER2 receptor or EGFreceptor may be inhibitied by administering to a patient a Formula I ADCwhich binds specifically to said growth factor receptor and achemotherapeutic agent wherein said antibody-drug conjugate and saidchemotherapeutic agent are each administered in amounts effective toinhibit growth of tumor cells in the patient.

A human patient susceptible to or diagnosed with a disordercharacterized by overexpression of ErbB2 receptor, may be treated byadministering a combination of a Formula I ADC and a chemotherapeuticagent. Such excessive activation may be attributable to overexpressionor increased production of the ErbB receptor or an ErbB ligand. In oneembodiment, a diagnostic or prognostic assay will be performed todetermine whether the patient's cancer is characterized by excessiveactivation of an ErbB receptor. For example, ErbB gene amplificationand/or overexpression of an ErbB receptor in the cancer may bedetermined. Various assays for determining suchamplification/overexpression are available in the art and include IHC,FISH and shed antigen assays.

Examples of cancer to be treated herein include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, gastrointestinal stromal tumor(GIST), pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The ADC formulation is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of ADC is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dosageregimen might range from about 1 μg/kg to 100 mg/kg or more, dependingon the factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. An exemplary dosingregimen comprises administering an initial loading dose of about 4mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theADC. Other dosage regimens may be useful.

Combination Therapy

An antibody-drug conjugate (ADC) may be combined in a pharmaceuticalcombination formulation, or dosing regimen as combination therapy, witha second compound having anti-cancer properties. The second compound ofthe pharmaceutical combination formulation or dosing regimen preferablyhas complementary activities to the ADC of the combination such thatthey do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, aromataseinhibitor, protein kinase inhibitor, lipid kinase inhibitor,anti-androgen, antisense oligonucleotide, ribozyme, gene therapyvaccine, anti-angiogenic agent and/or cardioprotectant. Such moleculesare suitably present in combination in amounts that are effective forthe purpose intended. A pharmaceutical composition containing an ADC mayalso have a therapeutically effective amount of a chemotherapeutic agentsuch as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNAbinder.

Alternatively, or additionally, the second compound may be an antibodywhich binds or blocks ligand activation of tumor-associated antigen orreceptor. The second antibody may be conjugated with a cytotoxic orchemotherapeutic agent, e.g., a macrocyclic depsipeptide, an auristatin,a calicheamicin, or a 1,8 bis-naphthalimide moiety. For example, it maybe desirable to further provide antibodies which bind to EGFR, ErbB2,ErbB3, ErbB4, or vascular endothelial factor (VEGF) in the oneformulation or dosing regimen.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein there is a time period while both (or all) activeagents simultaneously exert their biological activities.

In one embodiment, treatment with an ADC of the present inventioninvolves the combined administration of an anticancer agent identifiedherein, and one or more chemotherapeutic agents or growth inhibitoryagents. Preparation and dosing schedules for such chemotherapeuticagents may be used according to manufacturers's instructions or asdetermined empirically by the skilled practitioner. Preparation anddosing schedules for such chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992).

The ADC may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (EP 616812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated ishormone independent cancer, the patient may previously have beensubjected to anti-hormonal therapy and, after the cancer becomes hormoneindependent, the anti-ErbB2 antibody (and optionally other agents asdescribed herein) may be administered to the patient. It may bebeneficial to also coadminister a cardioprotectant (to prevent or reducemyocardial dysfunction associated with the therapy) or one or morecytokines to the patient. In addition to the above therapeutic regimes,the patient may be subjected to surgical removal of cancer cells and/orradiation therapy.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Metabolites of the Antibody-Drug Conjugates

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products may be identified by preparing a radiolabelled (e.g.¹⁴C or ³H) ADC, administering it parenterally in a detectable dose (e.g.greater than about 0.5 mg/kg) to an animal such as rat, mouse, guineapig, monkey, or to man, allowing sufficient time for metabolism to occur(typically about 30 seconds to 30 hours) and isolating its conversionproducts from the urine, blood or other biological samples. Theseproducts are easily isolated since they are labeled (others are isolatedby the use of antibodies capable of binding epitopes surviving in themetabolite). The metabolite structures are determined in conventionalfashion, e.g. by MS, LC/MS or NMR analysis. In general, analysis ofmetabolites is done in the same way as conventional drug metabolismstudies well-known to those skilled in the art. The conversion products,so long as they are not otherwise found in vivo, are useful indiagnostic assays for therapeutic dosing of the ADC compounds.

Metabolites include the products of in vivo cleavage of the ADC wherecleavage of any bond occurs that links the drug moiety to the antibody.Metabolic cleavage may thus result in the naked antibody, or an antibodyfragment. The antibody metabolite may be linked to a part, or all, ofthe linker. Metabolic cleavage may also result in the production a drugmoiety or part thereof. The drug moiety metabolite may be linked to apart, or all, of the linker.

Articles of Manufacture

In another embodiment, an article of manufacture, or “kit”, containingADC and materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, or blisterpack. The containers may be formed from a variety of materials such asglass or plastic. The container holds an antibody-drug conjugate (ADC)composition which is effective for treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anADC. The label or package insert indicates that the composition is usedfor treating the condition of choice, such as cancer.

In one embodiment, the article of manufacture may further comprise asecond (or third) container comprising a pharmaceutically-acceptablebuffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution, anda package insert indicating that the first and second compounds can beused to treat cancer. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EXAMPLES Example 1 Preparation of Cysteine Engineered Antibodies forConjugation by Reduction and Reoxidation

Full length, cysteine engineered monoclonal antibodies (ThioMabs) areexpressed in CHO cells and bear cysteine adducts (cystines) on theengineered cysteines due to cell culture conditions (US 2007/0092940;Junutula et al “CYSTEINE ENGINEERED ANTI-MUC16 ANTIBODIES AND ANTIBODYDRUG CONJUGATES”, U.S. Ser. No. 60/916,657, filed 8 May 2007). Toliberate the reactive thiol groups of the engineered cysteines, theThioMabs are dissolved in 500 mM sodium borate and 500 mM sodiumchloride at about pH 8.0 and reduced with about a 50-100 fold excess of1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al(1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.)for about 1-2 hrs at 37° C. The reduced ThioMab is diluted and loadedonto a HiTrap S column in 10 mM sodium acetate, pH 5, and eluted withPBS containing 0.3M sodium chloride. The eluted reduced ThioMab istreated with 2 mM dehydroascorbic acid (dhAA) at pH 7 for 3 hours, or 2mM aqueous copper sulfate (CuSO₄) at room temperature overnight. Ambientair oxidation may also be effective. The buffer is exchanged by elutionover Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Abvalue is checked by determining the reduced antibody concentration fromthe absorbance at 280 nm of the solution and the thiol concentration byreaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of theabsorbance at 412 nm.

Example 2 Conjugation of Cysteine Engineered Antibodies and Drug-LinkerReagents

After the reduction and reoxidation procedures of Example 1, thecysteine engineered antibody is dissolved in PBS (phosphate bufferedsaline) buffer and chilled on ice. About 1.5 molar equivalents, relativeto engineered cysteines per antibody, of nemorubicin metabolite oranalog drug-linker reagent, such as MC-D (maleimidocaproyl),MC-val-cit-PAB-D, or MC-val-cit-PAB-D, with a thiol-reactive functionalgroup such as maleimido, is dissolved in DMSO, diluted in acetonitrileand water, and added to the chilled reduced, reoxidized cysteineengineered antibody in PBS. After about one hour, an excess of maleimideis added to quench the reaction and cap any unreacted antibody thiolgroups. The reaction mixture is concentrated by centrifugalultrafiltration and the antibody-drug conjugate is purified and desaltedby elution through G25 resin in PBS, filtered through 0.2 μm filtersunder sterile conditions, and frozen for storage.

Example 3 Preparation of Ab-MC-PNU(159682) by Conjugation of Antibodyand MC-PNU(159682)

Antibody, dissolved in 500 mM sodium borate and 500 mM sodium chlorideat pH 8.0 is treated with an excess of 100 mM dithiothreitol (DTT).After incubation at 37° C. for about 30 minutes, the buffer is exchangedby elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA.The thiol/Ab value is checked by determining the reduced antibodyconcentration from the absorbance at 280 nm of the solution and thethiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) anddetermination of the absorbance at 412 nm. The reduced antibodydissolved in PBS is chilled on ice.

The drug linker reagent, maleimidocaproyl-PNU(159682), dissolved inDMSO, is diluted in acetonitrile and water at known concentration, andadded to the chilled reduced antibody in PBS. After about one hour, anexcess of maleimide is added to quench the reaction and cap anyunreacted antibody thiol groups. The reaction mixture is concentrated bycentrifugal ultrafiltration and Ab-MC-PNU(159682) is purified anddesalted by elution through G25 resin in PBS, filtered through 0.2 μmfilters under sterile conditions, and frozen for storage.

Example 4 Preparation of Ab-MCC-PNU(159682)

Purified antibody is derivatized with (succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, PierceBiotechnology, Inc) to introduce the MCC linker. Antibody is treated at20 mg/mL in 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA,pH 6.5 with 7.5 molar equivalents of SMCC (20 mM in DMSO, 6.7 mg/mL).After stirring for 2 hours under argon at ambient temperature, thereaction mixture is filtered through a Sephadex G25 column equilibratedwith 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5.Antibody containing fractions are pooled and assayed.

Ab-MCC from above is diluted with 50 mM potassium phosphate/50 mM sodiumchloride/2 mM EDTA, pH 6.5, to a final concentration of about 10 mg/ml,and reacted with a 10 mM solution of thiol-modified PNU(159682) (1.7equivalents assuming 5 MCC/Ab, 7.37 mg/ml) in dimethylacetamide. Thereaction is stirred at ambient temperature under argon 16.5 hours. Theconjugation reaction mixture is filtered through a Sephadex G25 gelfiltration column (1.5×4.9 cm) with 1×PBS at pH 6.5. The drug toantibody ratio (p) may be about 2 to 5, as measured by the absorbance at252 nm and at 280 nm.

Example 5 Preparation of Ab-SPP-PNU(159682)

Purified antibody is derivatized withN-succinimidyl-4-(2-pyridylthio)pentanoate to introduce dithiopyridylgroups and form Ab-SPP-Py. Purified antibody (376.0 mg, 8 mg/mL) in 44.7mL of 50 mM potassium phosphate buffer (pH 6.5) containing NaCl (50 mM)and EDTA (1 mM) is treated with SPP (5.3 molar equivalents in 2.3 mLethanol). After incubation for 90 minutes under argon at ambienttemperature, the reaction mixture is gel filtered through a Sephadex G25column equilibrated with 35 mM sodium citrate, 154 mM NaCl, 2 mM EDTA.Antibody containing fractions are pooled and assayed. The degree ofmodification of the antibody is determined as described above.

Ab-SPP-Py (about 10 tmoles of releasable 2-thiopyridine groups) isdiluted with the above 35 mM sodium citrate buffer, pH 6.5, to a finalconcentration of about 2.5 mg/mL. Thiol-modified PNU(159682) (1.7equivalents, 17 μmoles) in 3.0 mM dimethylacetamide (DMA, 3% v/v in thefinal reaction mixture) is then added to the antibody solution. Thereaction proceeds at ambient temperature under argon for about 20 hours.

The reaction is loaded on a Sephacryl S300 gel filtration column (5.0cm×90.0 cm, 1.77 L) equilibrated with 35 mM sodium citrate, 154 mM NaCl,pH 6.5. The flow rate may be about 5.0 mL/min and 65 fractions (20.0 mLeach) are collected. The number of drug molecules linked per antibodymolecule (p) is determined by measuring the absorbance at 252 nm and 280nm.

Example 6 Preparation of Ab-BMPEO-PNU(159682)

An antibody with a reactive cysteine thiol group, such as a cysteineengineered antibody or an antibody previously treated with a reducingagent such as DTT to reduce disulfide linkages, is reacted with1,11-bis-maleimidotriethyleneglycol (BM(PEO)3 (Pierce BioTechnology,ThermoScientific), leaving an unreacted maleimido group on the surfaceof the antibody. This may be accomplished by dissolving BM(PEO)3 in a50% ethanol/water mixture to a concentration of 10 mM and adding atenfold molar excess to a solution containing antibody in phosphatebuffered saline at a concentration of approximately 1.6 mg/ml (10micromolar) and allowing it to react for 1 hour to form antibody-linkerintermediate, Ab-PEO. Excess BM(PEO)3 is removed by gel filtration(HiTrap column, Pharmacia) in 30 mM citrate, pH 6 with 150 mM NaClbuffer. An approximate 10 fold molar excess thiol-modified PNU(159682)is dissolved in dimethyl acetamide (DMA) and added to the Ab-PEOintermediate. Dimethyl formamide (DMF) may also be employed to dissolvethe drug moiety reagent. The reaction mixture is allowed to reactovernight before gel filtration or dialysis into PBS to remove unreactedPNU(159682). Gel filtration on S200 columns in PBS is used to removehigh molecular weight aggregates and furnish purifiedAb-PEO-PNU(159682).

All patents, patent applications, and references cited throughout thespecification are expressly incorporated by reference.

We claim:
 1. A drug-linker reagent comprising the structure:

wherein X⁴ is a linker comprising a thiol reactive functional group selected from maleimide, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.
 2. The drug-linker reagent of claim 1 wherein X⁴ is a linker comprising maleimide.
 3. The drug-linker reagent of claim 1 wherein X⁴ is a linker comprising bromoacetamide.
 4. The drug-linker reagent of claim 1 wherein X⁴ is a linker comprising pyridyl disulfide. 